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1、C组轮状病毒主要基因全片段扩增及武汉地方株Wu82的VP6和VP7编码基因序列分析2007,23(12)中国人兽共患病ChineseJournalofZoonoses1167文章编号:1002—2694(2007)12—1167～05C组轮状病毒主要基因全片段扩增及武汉地方株Wu82的VP6和VP7编码基因序列分析*王远虹.,周璇,小林宣道.,朱泽荣,刘满清,杨占秋.摘要:目的建立c组轮状病毒VP6,VP7,VP4和NSP4基因全片段扩增方法;明确武汉地方株Wu82与其他毒株的系统发生关系.腹泻患者大便.方法PAGE筛查病毒;设计9对引物RT—PCR扩增VP6,VP7,VP4和NSP4基因;
2、VP6,VP7序列及亲水位点分析.结果Wu82VP6和VP7基因核酸序列与其它人C组轮状病毒相比同源性分别为98.45～97.12和99.34～94.83.结论C组轮状病毒基因非常保守,进化缓慢.2001年武汉地方株Wu82与1995年武汉地方株208株相比.VP7和VP6抗原表位存在差异.关键词:轮状病毒;C组;基因;序列测定;进化分析;亲水性分析中图分类号:R373.9文献标识码:AAmplificationoftheentirefragmentofgroupCrotavirusgenesandthesequenceanalysisonVP6andVP7genesinthefieldst
3、rainWu一82isolatedinWuhanareaWANGYuan-hong,ZHOUXuan,KOBAYASHINobumichi,ZHUZe-rong,UUMan-qing,YANGZhan-qiu(WuhanCentersforDiseasesPrevention&Contro1.Wuhan430022.China)ABSTRACT:ToestablishamethodtoamplifytheentirefragmentofgenesVP6,VP7,VP4andNSP4fromgroupCrota—virus.Inordertodefinethephylogeneticr
4、elationsamongthefieldstrainWu-82andotherviralstrains,4362stoolspecimensfrompatientswithdiarrheainallageswerecollectedduringtwoperiodsoftime,fromDec2000toApr2003andfromNov2003toFeb2007.ThepresenceofrotavirusesinStOOlspecimenswasdeterminedbydetectionofthedsRNAfragmentofrotavirusbypolyacrylamidegelele
5、ctrophoresis,andtheentirefragmentsofVP7,VP6,VP4andnsp4geneswereamplifiedbyRT—PCRwith9pairsofspecificprimers.NucleicacidsequencedataandhydrophilicityanalysisofthededucedaminoacidswereanalyzedusingtheGeneWorkssoftwarepackageandtheMEGA4program.ItwasdemonstratedthattheVP6andVP7genesfromW82strainwerequi
6、teconservedwithfewmutations,andthephylogeneticrelationoftheVP6genefromWu-82strainwasclosertotheNigerianstrainJajeriandSpanishstrainBCN6,whereastheVP7geneofthisstrainwasclosertoJapanesestrainE-93andColumbiastrainJaveriana.ThesequenceofthededucedaminoacidsofVP6andVP7ofWu一82strainwasdistinctfromthatof
7、the208strainisolatedinl995atWuhanarea.MostofthedifferentaminoacidsbetweenWu一82and208strainswasfoundtobelocatedinthehydrophilicityregions.ThehomologiesofnucleotidesequencesofVP6andVP7geneswithothergroupCro—t
